Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase.
Association and dissociation rate constants obtained by stopped-flow spectroscopy have permitted definition of a kinetic scheme for recombinant human dihydrofolate reductase that correctly predicts full time course kinetics of the enzymatic reaction over a wide range of substrate and product concentrations. The scheme is complex compared with that for the bacterial enzyme and involves branched pathways. It successfully accounts for observed rapid hysteresis preceding steady state and for the nonhyperbolic dependence of steady-state rate on substrate and product concentrations. The major branch point in the catalytic cycle occurs at E.NADP.H4folate because either NADP or H4folate can dissociate from the ternary product complex (koff = 84 s-1 and 46 s-1, respectively). The rate of conversion of enzyme-bound substrates to products is very fast (k = 1360 s-1) and nearly unidirectional (Kequ = 37) so that other steps limit the catalytic rate. At saturating substrate concentrations these steps include release of NADP and H4folate from E.NADP.H4folate and release of products from the two abortive complexes E.NADPH.H4folate (koff = 225 s-1) and E.NADP.H4folate (koff = 4.6 s-1). Since NADP dissociates slowly from E.NADP.H2folate nearly 90% of the enzyme accumulates as this complex at steady state. Nonetheless, the catalytic rate is maintained at 12 s-1 by rapid flux of a small portion of the enzyme through an alternate branch. At physiological concentrations of substrates and products the steady-state rate is limited primarily by the rate of H2folate binding to E.NADPH so that the enzyme is extremely efficient.
Appleman
J R
JR
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Beard
W A
WA
Delcamp
T J
TJ
Prendergast
N J
NJ
Freisheim
J H
JH
Blakley
R L
RL
D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013997:Time Factors
D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase